Cholesterol is the major steroid constituent of animal tissue, and is a normal component of plasma and essentially all cell membranes. Cholesterol is a hydrophobic hydrocarbon compound with a reactive hydroxyl group. It is a 3-hydroxy sterol (having a perhydro-1,2-cyclopentenophenanthrene ring system skeleton) with an aliphatic side chain at the C-17 position. In theory, cholesterol plays a major role in regulating the fluidity and permeability of the cell membrane by forming an intercalated structure among the membrane phospholipids.
Cholesterol can be obtained from the diet or synthesized in the liver. It can also be manufactured within the cell, if needed, but in practice most cells receive their cholesterol externally. Cholesterol synthesis occurs according to an enzyme-mediated biosynthetic pathway, in which the rate limiting enzyme is believed to be hydroxymethylglutaryl-Co-A reductase ("HMG CoA reductase") See, for example, Javitt, U.S. Pat. No. 4,427,668. This enzyme catalyzes the formation of mevalonic acid, a cholesterol precursor, from hydroxymethyl glutaric acid. Cholesterol production can be regulated, in theory, by interfering with HMG CoA reductase. Goodman & Gilman, "The Pharmacological Basis of Therapeutics," 7th Ed., MacMillan (New York: 1985), pp. 841-843; Kandustsch et al., Science, 201, 498 (1978).
The excessive accumulation of cholesterol has been implicated as the prime causative factor in a number of diseases. In particular, elevated concentrations of cholesterol can cause and/or hasten atherosclerosis, which is characterized by an abnormal hardening and thickening of the arterial walls due to the accumulation and deposition of fatty materials, including cholesterol. This, in turn, can lead to thrombosis and infarction. For a review of cholesterol-mediated pathologies, see, "Hormones and Metabolic Control," D. A. White, B. Middleton, and M. Baxter, editors; Edward Arnold Publisher; London, 1984, pages 73-90.
The drugs currently in use for the inhibition of cholesterol synthesis have an impact primarily on the liver, and are believed to function by causing upregulation of the LDL receptors of the liver to increase liver uptake of cholesterol and decrease serum levels or by inhibiting HMG CoA reductase, so that cholesterol production is reduced.
Representative of known drugs that are currently in use include mevinolin and cholestyramine, which cause the upregulation of LDL receptors in the liver. Mevinolin, which is an allosteric inhibitor of HMG CoA reductase, achieves this regulation effect by inhibiting cholesterol synthesis in the liver. This stimulates increased LDL receptor activity in the liver, causing hepatic uptake of cholesterol, which in turn reduces plasma cholesterol. Cholestyramine increases the need for bile acid synthesis from cholesterol in the liver, which in some individuals will result in upregulation of LDL receptors. Once again, this reduces plasma cholesterol. The primary effect of both compounds is therefore limited to the liver. The known compounds are not intended to and do not significantly effect cells outside the liver. A strategy targeted for the liver is effective using the known compounds because, unlike other cells, liver cells can uniquely metabolize (and remove) cholesterol to bile acids. Indeed, compounds such a mevinolin would have a detrimental effect on the accumulation of tissue cholesterol if targeted on cells outside the liver, since the compound would upregulate the LDL receptors and would cause rather than reduce cholesterol accumulation.
Another known compound is 26-hydroxycholesterol which is the subject of U.S. Pat. No. 4,427,668, the disclosure of which is hereby incorporated by reference. See, also Javitt et al., (1981) J.Biol.Chem, Vol. 256(24), pages 12644-12646; Brooks et al., (1983) Biochem Soc. Trans., Vol. 11, pages 700-701; Koopman et al., (1987) J. Chromatogr., Vol. 416, pages 1-13; Esterman et al., (1983) J. Lipid Res., Vol. 24, pages 1304-1309; and Taylor et al., (1984) J. Biol. Chem., Vol. 259(20), pages 12382-12387.
26-hydroxycholesterol is a bile acid intermediate and is believed to have a role in modulating cellular cholesterol homeostasis. For a review of the biological effects of 26 -hydroxy-cholesterol, see, Javitt, N. B. (1990) J. Lipid Res., Vol. 31, pages 1527-1533. Synthetic methods for preparing 26-hydroxy-cholesterol have been reported in the literature. See, for examples, Tschesche et al. (1979) Chem Ber., Vol. 112, pages 2680-2691 and Seo et al. (1986) J. Chem. Soc. Perkin Trans. I, pages 411-416. The described methods generally involve harsh multi-step reactions and produce low yields of the hydroxysteroid.
26-Aminocholesterol was described to be a potent downregulator of LDL receptor and inhibitor of cholesterol biosynthesis in vitro, having equal or greater biological potency than the hydroxyl intermediate. See, Miao et al. (1988) Biochem. J., Vol. 255, pages 1049-1052 and U.S. Pat. No. 4,939,134 (the '134 patent), the disclosures of which are hereby incorporated by reference. This compound is known to selectively inhibit and reduce LDL binding in human fibroblast cells, while having little or no effect on human hepatoma (HepG2) cells. In addition, the compound was also found to downregulate cholesterol synthesis by non-hepatic cells.
Synthetic methods for preparing (25R)-26-aminocholesterol have been reported. Miao et al. (1988) and the '134 patent, for example, describe the preparation of 26-aminocholesterol by a lithium aluminum hydride reduction of 26-azidocholesterol 3-acetate. Tschesche et al. (1979) Chem. Ber., Vol. 112, pages 2680-2691, describes preparation of 26-phthalimidocholesterol and its basic hydrolytic conversion to (25R)-26-aminocholesterol. Preparation of the aminosteroid by these methods, however, requires 26-hydroxycholesterol as starting material, uses complicated protection/deprotection reactions, and results in low yields of the desired product.
Klimova et al., (1982) Khim. Pharm. Zh., Vol. 16, page 451, describes preparation of 26-acetylamino analogs of cholesterol from solasodine, a plant alkaloid and amino analog of diosgenin. According to the described procedure, 3.beta.-acetoxy-26-acetylamino-16,22-dione, derived from oxidation of O,N-diacetylated solasodine, is subjected to a Clemmenson reduction using activated zinc amalgam which produces a mixture of the diacetylated derivatives of 26-aminocholest-5-ene-22-one as a major product and 26-aminocholesterol as a by-product. Because 26-aminocholesterol is produced in low yields, the disclosed reaction is not useful in preparing this aminosteroid. Until now, a simple method for preparing 26-aminocholesterol in high yields has not been available.
It has now unexpectedly been discovered that (25R)-26-aminocholesterol can be prepared directly from simple intermediates, in high yields, and which avoids the need of 26-hydroxycholesterol as starting material and the use of protective group chemistry.